Descriptive and Molecular Epidemiology of Antibiotic-Resistant Gram-Negative Enteric Bacteria from Dairy Cattle
Open Access
- Author:
- Sawant, Ashish Anant
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 03, 2005
- Committee Members:
- Bhushan M Jayarao, Committee Chair/Co-Chair
Avery August, Committee Member
Brenda Love, Committee Member
Lorraine Sordillo, Committee Member
Stephen John Knabel, Committee Member
Jeffrey Watts, Committee Member - Keywords:
- molecular epidemiology
dairy cattle
enteric bacteria
antibiotic resistance - Abstract:
- A survey was conducted to study the antibiotic usage on dairy herds (n=113)in Pennsylvania. The findings of the study showed that antibiotics were extensively used on dairy herds for therapeutic and prophylactic purposes. Beta-lactams and tetracyclines were the most widely used antibiotics on dairy herds. Beta-lactams were used for clinical mastitis, dry cow therapy, metritis, and pneumonia; while tetracycline was primarily used in medicated milk replacers. Extralabel use of antibiotics was practiced on many farms. Extralabel use of a third generation cephalosporin, Ceftiofur was reported by 18% of the dairy herds surveyed. Recommended prudent practices of antibiotic usage (record keeping, written treatment plans, following labeled instructions, and veterinarian's advice on antibiotic usage) were not widely practiced. It is felt that current practices related to the antimicrobial usage on farms could contribute to the development of antibiotic resistant bacteria. Lactating dairy cattle (n=313) on 33 dairy herds were examined for antibiotic resistant gram-negative enteric bacteria. Gram-negative enteric bacteria resistant to ampicillin, oxytetracycline, florfenicol, spectinomycin, and neomycin were isolated from feces of 31, 31, 7, 5, and 1% of lactating cows, respectively. Gram-negative enteric bacteria resistant to ampicillin, florfenicol, neomycin, tetracycline, and spectinomycin accounted for 9, 5, 1, 14, and 10% of the total Gram-negative enteric bacteria. Gram-negative enteric bacteria belonging to 12 species were isolated from dairy cattle, of which Escherichia coli (87%) was the most predominant species. Monte Carlo analysis revealed that E. coli would require 126 days to undergo a 3 log reduction when held in sterile water at 7oC. It was observed that dairy producers that fed medicated milk replacers to calves were 3.4 fold more likely to have lactating cattle shed tetracycline-resistant Gram-negative enteric bacteria. However a similar relationship was not observed with calves. Ampicillin- and tetracycline-resistant E. coli were the predominant isolates among the gram-negative species isolated and these isolates were genetically diverse. The findings of the study suggest that resistant Gram-negative enteric microflora was widely prevalent on farms. Tetracycline determinants tet(B) and tet(A) were detected in 93 and 7% of the isolates, respectively. These tet determinants were located on the chromosome, and sequence analysis revealed association of tetR and tetA with transposon Tn10. This is the first report of a chromosomally located tet efflux pump associated with Tn10 in enteric E. coli isolated from lactating cattle. The results of this study show that one of the pathways through which tetracycline resistance can be mediated is through transposable element Tn10 that harbor a tet(B) determinant. Ampicillin resistant enteric E. coli (n=94 isolates) from lactating cattle were examined for susceptibility to other antibiotics and genetic determinants that encode for beta-lactam resistance. The majority of the E. coli isolates were resistant to tetracycline (88%) followed by chloramphenicol (30%), spectinomycin (35%), ticarcillin (33%), ticarcillin/ clavulanic acid (23%) and ceftiofur (27%). Multidrug resistance (>3 to 8 antimicrobial agents) was observed in 44 of 94 (47%) isolates, interestingly on most occasions multidrug resistant ceftiofur isolates were also resistant to chloramphenicol. All E. coli isolates were susceptible to the 4th generation cephalosporin cefepime. The extended-spectrum beta-lactamase enzymes were not detected in ceftiofur resistant isolates. Ampicillin-resistant isolates contained blaTEM, while ceftiofur-resistant isolates contained cephamycinase (blaCMY). Class I integrons of >1 kb were observed in 10 of 75 E. coli isolates. Sequence analysis revealed that the integrons encoded for streptomycin and spectinomycin (aadA), and/or trimethoprim (dfr) resistance. These findings suggest commensal enteric E. coli and other Gram-negative enteric bacteria from lactating cattle can be a significant reservoir of antibiotic-resistant determinants. Widespread prevalence of antibiotic-resistant E. coli could also pose a risk to public health as these organisms have the ability to survive for long periods in the environment and could gain access to the food chain.